Ghrelin signaling in dCA1 suppresses neuronal excitability and impairs memory acquisition via PI3K/Akt/GSK-3ß cascades.

Ghrelin is a circulating peptide hormone that promotes feeding and regulates metabolism in humans and rodents. The action of ghrelin is mediated by the growth hormone secretagogue receptor type 1a (GHSR-1a) that is widely distributed in the brain, including the hippocampus. Studies have demonstrated the critical role of hippocampal ghrelin/GHS-R1a signaling in synaptic physiology and memory. However, those findings are controversial, and the mechanism underlying ghrelin modulation of learning and memory is uncertain. Here, we report that micro-infusion of ghrelin in the CA1 region of the dorsal hippocampus during training specifically impairs memory acquisition. The activation of GHS-R1a and the subsequent PI3K/Akt/GSK3ß signaling cascades are involved in this process. Moreover, we report that bath application of ghrelin suppresses the intrinsic excitability of dCA1 pyramidal neurons through activating GHS-R1a, and PI3K inhibitor LY294002 blocks ghrelin's effect. However, LY294002 fails to rescue ghrelin-induced LTP impairment. Our findings support an adverse effect of ghrelin-dependent activation of GHS-R1a on memory acquisition, and suggest that PI3K/Akt/GSK3ß signaling-dependent repression of neuronal intrinsic excitability is an important novel mechanism underlying memory inhibition of ghrelin in the hippocampus.

The effect of obestatin on anxiety-like behaviour in mice.

Obestatin is a 23 amino acid-peptide, derived from the same preproghrelin-gene as ghrelin. Obestatin was originally reported as a ghrelin antagonist with anorexigenic activity, but later it was proven to be involved in multiple processes including sleep, memory retention, anxiety, morphine-induced analgesia and withdrawal. In the present study, in male CFLP mice, by using computerised open field (OF) and elevated plus maze (EPM) tests we have investigated the behavioural effects of the acute intracerebroventricular (icv) administration of obestatin alone, and following ghrelin receptor blockage with [d-Lys3]-Growth Hormone Releasing Peptide-6 ([d-Lys3]- GHRP6) or corticotropin-releasing hormone (CRH) receptor 1 antagonism with antalarmin. Plasma corticosterone levels were measured for each treatment group by using chemofluorescent assay. Our results in the EPM test showed that obestatin reduced the percent of time spent in the open arms. The basal locomotor activity (ambulation distance and time, rearing and jumping) was not influenced significantly neither in the obestatin-treated groups, nor in those receiving pre-treatment with antalarmin or [d-Lys3]-GHRP6. The percentage of central ambulation distance however was decreased by obestatin, while the percentage of time spent in the central zone showed a decreasing tendency. The administration of antalarmin or [d-Lys3]-GHRP6 have both reversed the effect of obestatin on central ambulation. Plasma corticosterone levels were elevated by obestatin, which effect was antagonised by the injection of antalarmin. These are the first results to indicate that obestatin exerts anxiogenic-like effect in mice, which might be mediated through ghrelin receptor and CRH activation.

Effects of ghrelin on the structural complexity of exocrine pancreas tissue architecture.

Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the potential effects of ghrelin on the morphology of exocrine pancreas (EP) remain unknown. In this work, using fractal analysis, we demonstrate that centrally administered ghrelin increases structural complexity and tissue disorder in rat EP. The study was carried out on a total of 40 male Wistar rats divided into four groups (n = 10): ghrelin-treated animals (average age, 1.5 months), ghrelin-treated animals (8.5 months), and controls (1.5 and 8.5 months). The pancreas tissue sections were stained with hematoxylin/eosin and visualized by light microscopy. For each animal, the average values of tissue fractal dimension, lacunarity, as well as parameters of co-occurrence matrix texture, were determined using tissue digital micrographs. The results indicate that ghrelin administration increases EP fractal dimension and textural entropy, and decreases lacunarity, regardless of the age. To our knowledge, this is the first study to investigate the effects of ghrelin on the morphological properties of pancreatic tissue, and also the first to apply fractal and textural analysis methods in quantification of EP tissue architecture.

Effect of ghrelin and anamorelin (ONO-7643), a selective ghrelin receptor agonist, on tumor growth in a lung cancer mouse xenograft model.

PURPOSE: Anamorelin (ONO-7643) is an orally active ghrelin receptor agonist in development for non-small cell lung cancer (NSCLC)-related anorexia/cachexia. It displays both orexigenic and anabolic properties via ghrelin mimetic activity and transient increases in growth hormone (GH). However, increasing GH and insulin-like growth factor-1 in cancer patients raises concerns of potentially stimulating tumor growth. Therefore, we investigated the effect of ghrelin and anamorelin on tumor growth in a murine NSCLC xenograft model. METHODS: Female nude mice (15-21/group) with established A549 tumors were administered ghrelin (2 mg/kg i.p.), anamorelin (3, 10, or 30 mg/kg p.o.), or vehicle controls daily for 28 days. Tumor growth, food consumption, and body weight were monitored. Murine growth hormone (mGH) and murine insulin-like growth factor-1 (mIGF-1) were measured in plasma. RESULTS: Tumor growth progressed throughout the study, with no significant differences between treatment groups. Daily food consumption was also relatively unchanged, while the percentage of mean body weight gain at the end of treatment was significantly increased in animals administered 10 and 30 mg/kg compared with controls (p < 0.01). Peak mGH levels were significantly higher in ghrelin- and anamorelin-treated animals than in controls, while peak mIGF-1 levels were slightly elevated but not statistically significant. All regimens were well tolerated. CONCLUSIONS: These findings demonstrate that neither anamorelin nor ghrelin promoted tumor growth in this model, despite increased levels of mGH and a trend of increased mIGF-1. Together with anamorelin's ability to increase body weight, these results support the clinical development of ghrelin receptor agonist treatments for managing NSCLC-related anorexia/cachexia.

Intracerebroventricular administration of metformin inhibits ghrelin-induced Hypothalamic AMP-kinase signalling and food intake.

BACKGROUND/AIMS: The antihyperglycaemic drug metformin reduces food consumption through mechanisms that are not fully elucidated. The present study investigated the effects of intracerebroventricular administration of metformin on food intake and hypothalamic appetite-regulating signalling pathways induced by the orexigenic peptide ghrelin. METHODS: Rats were injected intracerebroventricularly with ghrelin (5 µg), metformin (50, 100 or 200 µg), 5-amino-imidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, 25 µg) and L-leucine (1 µg) in different combinations. Food intake was monitored during the next 4 h. Hypothalamic activation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), regulatory-associated protein of mTOR (Raptor), mammalian target of rapamycin (mTOR) and p70 S6 kinase 1 (S6K) after 1 h of treatment was analysed by immunoblotting. RESULTS: Metformin suppressed the increase in food consumption induced by intracerebroventricular ghrelin in a dose-dependent manner. Ghrelin increased phosphorylation of hypothalamic AMPK and its targets ACC and Raptor, which was associated with the reduced phosphorylation of mTOR. The mTOR substrate, S6K, was activated by intracerebroventricular ghrelin despite the inhibition of mTOR. Metformin treatment blocked ghrelin-induced activation of hypothalamic AMPK/ACC/Raptor and restored mTOR activity without affecting S6K phosphorylation. Metformin also reduced food consumption induced by the AMPK activator AICAR while the ghrelin-triggered food intake was inhibited by the mTOR activator L-leucine. CONCLUSION: Metformin could reduce food intake by preventing ghrelin-induced AMPK signalling and mTOR inhibition in the hypotalamus.